Immunogenic muc1 glycopeptides

ABSTRACT

Provided are novel MUC1 peptides for use in anti-tumor vaccination and methods of producing those peptides. Furthermore, methods of producing a population of autologous antigen presenting cells (APCs) and of producing genetically engineered APCs, which are capable of inducing effective immune responses against MUC1 are described. The described peptides are particularly useful for the treatment of breast cancer or other MUC1-positive carcinomas including colorectal, pancreatic and gastric carcinomas.

FIELD OF THE INVENTION

The present invention relates to MUC1 peptides and to methods ofproducing those peptides. The invention further relates to an exvivo-method of producing a population of autologous antigen presentingcells (APCs) and of producing genetically engineered APCs, which arecapable of inducing effective immune responses against MUC1. Theinvention also relates to APCs, which are obtainable by these methods aswell as to the use of the above mentioned peptides and APCs in apharmaceutical composition for the treatment of breast cancer or otherMUC1-positive carcinomas including colorectal, pancreatic and gastriccarcinomas.

BACKGROUND OF THE INVENTION

MUC1 is overexpressed in breast cancer and by many other carcinomas andthe tumor-associated glycoform of the mucin is known to expose multiplepeptide epitopes within its repeat domain. These immunogenic peptideepitopes make MUC1 a promising tumor antigen with diagnostic as well astherapeutic potential in the treatment of cancer.

The development of effective vaccine and immunotherapies for humancancers and infectious agents often is dependent on the generation ofprotective immune responses to specific domains of membrane proteins.The tandem repeat (TR) domain of the breast, pancreatic, and ovariantumor antigen, human mucin MUC1 (Barnd et al., PNAS USA 86 (1989),7159-7163; Jerome et al., Cancer Res. 51 (1991), 2908-2916), theprincipal neutralizing domain of HIV-1 (Javaherian et al., PNAS USA 86(1989), 6768-6772; Javaherian et al., Science 250 (1990), 1590-1593) andthe proline rich neutralization domain of the feline leukemia virusexternal surface unit protein (gp-70) (Nunberg et al., PNAS 81 (1984),3675-3679; Elder et al., J. Virol. 61 (1987), 8-15; Strouss et al., J.Virol. 61 (1987), 3410-3415; Nick et al., J. Gen. Virol. 71 (1990),77-83) are examples hereof.

Regarding MUC1, humoral and cellular responses have been demonstrated incancer patients (Kotera et al., Cancer Res. 54 (1994), 2856-2860; Barndet al., Proc. Natl. Acad. Sci. USA 86 (1989), 7159-7.163), but also inpregnant woman (Hilkens et al., Cancer Res. 46 (1986), 2582-2587) andhealthy individuals (Agrawal et al., Cancer Res. 55 (1995), 2257-2261).Although these natural responses are usually insufficient to fight theprogress of cancer, MUC1-derived peptides or glycopeptides are usedcurrently in clinical trials to trigger therapeutically andprophylactically immune reactions in humans (Karanikas et al., J. Clin.Invest. 100 (1997), 2783-2792; Goydos et al., J. Surg. Res. 63 (1996),298-304).

There is growing evidence that triggering of efficient humoral and CTLresponses to MUC1 needs the activation of specific T helper cell clones,which is induced by MHC class II-presented antigen fragments. Thegeneration of MHC class II-restricted peptide epitopes by antigenpresenting cells (APCs) like dendritic cells (DCs) follows a multistepprocess starting with endocytosis, followed by the processing in lateendosomal compartments and resulting in the binding of proteolyticpeptide fragments to MHC class II proteins and their transport to thecell surface. While many aspects of this complex process have beenelucidated there is currently little evidence on the processing and MHCclass II presentation of glycosylated antigens, in particular of thehighly O-glycosylated mucin antigens. To enable the design of efficienttumor vaccines on the basis of MUC1 knowledge on how DCs or other APCsdeal with O-glycosylated peptides is of importance. One particularquestion in this context refers to the fate of complex O-linked glycansduring processing, since efficient peptide fragmentation may affordcomplete or partial removal of sugars prior to proteolysis. O-linkedglycans could also direct the processing with respect to theaccessibility of cleavage sites and hence restrict the pattern ofpeptide fragments on the one hand, while they enrich the pattern ofepitopes on the other.

Although several of the cathepsins have been identified as components ofthe processing machinery (Honey et al., J. Biol. Chem. 276 (2001),22573-22578; Shen et al., J. Immunol. 158 (1997), 2723-2730), it iscurrently not known which enzyme(s) are involved in the processing ofMUC1 and at which sites within the repeat domain they actually cleavethe protein. Expectedly, there are multiple cleavage sites and onlysubfractions of the generated peptide fragments may fulfil therequirements for binding to MHC class II molecules. Thus, while there isa constant need of specific and immunogenic MUC1 peptides for use asanti-cancer vaccines, so far the structural requirements for designingimmunogenic MUC1 peptides had not been elucidated.

The solution to said technical problem is achieved by providing theembodiments characterized in the claims, and described further below.

SUMMARY OF THE INVENTION

The present invention is directed to novel immunogenic MUC1 peptides,which can be used for immunization in mammals, especially in humans. Inparticular, peptides of least 9 amino acids in length derived from thetandem repeat domain of MUC1 and having the amino acid sequence SAP atits N-terminus are provided.

The present invention also concerns nucleic acids encoding such peptidesand vectors comprising said nucleic acids as well as host cellstransfected with nucleic acids or vectors of the invention.

Furthermore, the present invention relates to a method of producing animmunogenic MUC1 peptide, which allows the originally containedglycosylation pattern to be conserved during the production process.

The use of the MUC1 peptides in accordance with the present inventionmay be accompanied by the use of further therapeutic agents such astoxins and anti-cancer drugs commonly used in the therapy or diagnosisof cancer.

It is another object of the present invention to provide a fusionmolecule comprising the peptide of the invention and a functional moietysuch as a toxin, label, etc.

It is another object of the present invention to provide a method ofproducing a population of autologous antigen presenting cells (APCs),which are capable of inducing effective immune responses against MUC1,comprising the steps of

-   (a) providing autologous APCs from a tumor patient;-   (b) contacting the autologous APCs from the tumor patient with an    effective amount of a peptide or fusion molecule of the invention    under conditions which allow endocytosis, processing and MHC class    II presentation of the peptide fragments by said APCs; and-   (c) isolating said peptide presenting APCs for the purpose of    immunotherapeutic application in the patient.

It is another object of the present invention to provide a method ofproducing genetically engineered APCs, which are capable of inducingeffective immune responses against MUC1, comprising the steps of

-   (a) providing a nucleic acid encoding at least one of the peptides    of the invention or a fusion molecule comprising said at least one    peptide;-   (b) transfecting the APCs with said nucleic acid; and-   (c) selecting APCs, which present said peptides in an MHC II    restricted manner.    APCs obtainable by said method are subject of the present invention    as well.

The peptides, fusion molecules, nucleic acids, vectors, APCs, andcompositions containing any one of those compounds can be used asvaccine, for example for the prevention and therapeutic treatment ofMUC1-positive carcinomas such as breast, colorectal, pancreatic andgastric cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: MUC repeat peptide processing by human dendritic cells. Solubleantigen, a 100mer peptide with free amino and carboxy termini andcorresponding to five repeats of the MUC1 repeat domain (HGV100), wasused for pulsing of human immature dendritic cells prepared fromperipheral blood monocytes. During pulsing the cells were simultaneouslymatured by induction with TNFα and anti CD40. After 24 h pulsing andmaturation the cell supernatant was run over a solid-phase extractioncolumn to isolate the peptide fragments. MALDI mass spectrometry in thepositive ion mode revealed the formation of SAP17, GVT20, GVT23, andSTA27 as the major cleavage products in the mass range from 1 to 3 kDa.Mass signal indicated by * represent peptide background not related toMUC1 antigen peptide.

FIG. 2: MUC1 glycopeptide processing by mouse dendritic cells.Bead-conjugated antigens, a mixture of biotinylated glycopeptides H1 toH3, SEQ ID NO: 5, (AHGVTSAPDTRPAPGSTAPPA) and H4 to H6(AHGVTSAPESRPAPGSTAPAA), SEQ ID NO: 6, corresponding to a partialsequence of the MUC1 tandem repeat domain and glycosylated with GalNAcat Thr5 (H1, H4), Thr10/Ser10 (H2, H5) or Thr17 (H3, H6), was used forpulsing of mouse dendritic cells DC2.4. Processing products wereaffinity-isolated from cellular fractions or from culture supernatantsby binding to streptavidin/polystyrene-coated beads, reduced withdithiothreitol to cleave the biotin label, and analysed by reflectronMALDI mass spectrometry in the positive ion mode. A, cellular fraction;B, cell culture supernatant; C, interpretation of mass spectrometricdata. The major signals at m/z 2249.0 (H1 to H3), SEQ ID NO: 5, and2223.0 (H4 to H6), SEQ ID NO: 6, correspond to the thiopropylatedprecursor glycopeptides, the signals at m/z 1695.7 (P1; SEQ ID NO: 7)and 1669.7 (P2; SEQ ID NO: 8) to the SAP16 fragments (P1 derived from H1to H3; P2 from H4 to H6), which bind non-specifically to thepolystyrene-coated bead surface.

FIG. 3: Peptide sequencing of processing products P1 and P2 by LC-MS/MSanalysis on a Qtof2 electrospray mass spectrometer. Processing productsin cellular supernatants from antigen-pulsed mouse DCs were separated bynanoflow liquid chromatography on a reversed-phase microcapillary columnand analysed online by electrospray mass spectrometry in the positiveion mode. B-ion and y-ion fragment series from the N-terminal andC-terminal sequences of the major peptide products from endopeptidasecleavage were assigned after deconvolution of the spectrum (A; P1 at m/z1695; B, P2 at m/z 1669) and were used to confirm the sequence of SAP16glycopeptides derived from N-biotinylated H1 to H6 glycopeptide antigens(refer to C).

FIG. 4: In vitro proteolysis of MUC1 glycopeptide A3 by human cathepsinL. N-terminally free or biotinylated MUC1 glycopeptide A3 (10 μg) weretreated for 3 h with 1 milliunit of cathepsin L in the presence orabsence of the cathepsin L/B-specific cysteine protease inhibitorZ-Leu-Leu-Leu-fluoromethyl ketone (1 μM) using 0.1M sodium acetate, pH5.5, containing 1 mM EDTA, and 1 mM DTT as reaction buffer.

Reflectron MALDI mass spectra were recorded in the positive ion modeusing α-cyano-4-hydroxycinnamic acid as matrix. A, N-terminally freeglycopeptide A3 in the absence of protease inhibitor (m/z 1857.7: SAP16;m/z 2324.0: A3 glycopeptide; Signals at m/z 1958.8 and 2115.8 correspondto products of a aminopeptidase contained in the human cathepsin Lpreparation); B, N-terminally free glycopeptide A3 in the presence ofprotease inhibitor; C, glycopeptide A3 N-terminally biotinylated withbiotin N-hydroxysuccinimide ester (Sigma) at the amino terminus to blockaminopeptidase activity (in the absence of protease inhibitor); (m/z1858.6: SAP16; m/z 2549.8: biotinylated A3 glycopeptide); D,glycopeptide A3 N-terminally biotinylated with biotinN-hydroxysuccinimide ester at the amino terminus (in the presence ofprotease inhibitor).

FIG. 5: Cathepsin L-like activity in low-density endosomes from mousedendritic cells cleaves MUC1 repeats at Thr-Ser: Low-density endosomesin mouse dendritic cells were separated from lysosomes and plasmamembranes by density gradient centrifigation in percoll/sucrose (30 ml).

A profile of β-hexosaminidase activity in the gradient fractionsdemonstrates colocalisation of the lysosomal marker enzyme in highdensity fractions. The insert shows identification of cathepsin L in awesternblot of gradient fractions and human cathepsin L as a positivecontrol. Fractions of 1 ml were collected and 20 μl samples were loadedonto 7.5% polyacrylamid gels. After SDS gelelectrophoresis the proteinswere blotted onto nitrocelulose membranes and stained for the presenceof cathepsin L using the monoclonal mouse antibody CPLH 3G10 defining aC-terminal peptide of murine and human mature enzyme (AlexisDeutschland, Grünberg, Germany).

FIG. 6: Proposed pathways of the cathepsin L-mediated processing of MUC1tandem repeat peptide and its control by O-glycosylation. Filled arrowsindicate cleavage sites of cathepsin L. Thin arrows indicate theformation of major (continuous lines) or minor fragmentation routes(dashed lines). GalNAc residues are marked by grey shaded rhombs, Galresidues by open circles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to immunogenic MUC1 peptides, which can beused for immunization in mammals, especially in humans. In particular,those peptides are convenient in size, i.e. they comprise or consist ofat least 9 consecutive amino acids derived from the tandem repeat domainof MUC1 and having the amino acid sequence SAP at their N-terminus.

The present invention is based on the observation that cathepsin L or aclosely related enzyme shows a very restricted fragmentation patternduring human and mouse DC processing with only two preferred cleavagesite per MUC1 repeat. Without intending to be bound by theory it isbelieved that the cleavage specificity and specific inhibition of theprotease were in agreement with the assumption that cathepsin L or aclosely related enzyme (cathepsins B or S) were involved in this highlyspecific cleavage.

The experimental set-up used biotinylated and non-tagged beads, coatedwith synthetic glycopeptides comprising one or more repeat units of MUCwith single or multiple O-linked core-type glycans. Exogenouslyadministered MUC1 peptide fragments were rapidly taken up by mousedendritic cells (DCs) and a large proportion was processed in lateendosomal compartments within 4 h. MUC1 repeat peptide derivedproteolytic fragments that were identified and sequenced show that theglycans are not removed during antigen processing and that the presenceof carbohydrates affects the cleavage sites yielding a differentrepertoire of cleaved peptides.

Surprisingly, the proteolytic products suggest a highly specificprocessing of the repeat peptide with one preferential cleavage site atthe Thr-Ser peptide bond. While human cathepsin D was unable to cleavethe MUC1 repeat peptide in vitro, human cathepsin L digestion resultedin specific hydrolysis of the Thr-Ser peptide bond. Since MUC1 sequencescontain a VTSA motif in every repeat unit, the generated fragments startwith the amino acid sequence SAP at their N-terminus. Furthermore, itturned out that cathepsin L cleaves the MUC1 repeat peptide at anadditional site, namely at His-Gly. Thus, intermediate products arisefrom the processing of GVT-20 fragments (see for example SEQ ID NO: 12)that are transformed into SAP17 fragments by a further proteolyticcleavage depending on the site-specific O-glycosylation.

Information on the structure of processed MUC1 glycopeptides is ofutmost importance for the design of tumor vaccines. IntactO-glycosylation on processed MUC1 repeat peptide contributes to agreater variety of the MHC class II-restricted helper T cell responses,thereby enhancing an overall anti-tumor response.

Hence, according to the invention, a peptide of least 9 amino acids inlength derived from the tandem repeat domain of MUC1 and having theamino acid sequence SAP at its N-terminus is provided.

The amino and nucleic acid sequences of human MUC1 are known and can befound, for example, in the SWISS PROT and GenBank database; see, e.g.,accession nos. NP_(—)877418 and NM_(—)182741.1 and references citedtherein. The MUC1 protein contains varying numbers of amino acids due toa length polymorphism resulting from individually variable repeatnumbers, and, in the moment, at least 9 isoforms are known (1/A, 2/B,3/C, 4/D, 5/SEC, 6/X, 7/Y, 8/Z and 9/S, which are produced byalternative splicing).

In this invention, specific peptides of MUC1 are contemplated, which arederived from a synthetic or natural MUC1 sequence, which has beencleaved enzymatically at the VTSA motif contained in all MUC1 sequences(or was chemically synthesized in case of synthetic fragments). In oneembodiment, the peptides of the present invention thus can be obtainedby cleavage of MUC1 sequences with cathepsin-L. Irrespective of thestarting amino acid position in the repeat sequence (TAP, AHG, GST) andof the length of the peptides (20mer, 21mer, 25mer, 100mer), cathepsin Lcleaves specifically between Thr-Ser in the VTSA motif of the repeatpeptide, thereby resulting in the peptides according to the invention.It is an essential feature of the present invention that all peptideshave the amino acid sequence SAP at or near their N-terminus. The mostimportant feature of the peptides of the invention is that they consistof or comprise at least one tandem repeat domain of at least 9 aminoacids as shown below for the peptides of SEQ ID NOS: 1 to 4 and 11, witha minimum tandem repeat sequence of 9 amino acids, e.g. from position 1to 9 of any one of SEQ ID NOS: 1 to 4 and 11. This also means that theamino acid sequence SAP does not need to be immediately at theN-terminus but may be preceded by one or more amino acids, for examplewith the amino acid sequence GVT with or without an additional aminoacid such as H, see, e.g., peptide fragments shown in FIGS. 1 and 6.However, peptides consisting or comprising said tandem repeat domainwith N-terminal deletions of one or more amino acids, even of the SAPmotif, are encompassed in the scope of the present invention as well, inparticular if those peptide variants exhibit substantially the sameimmunological and/or biological activity as a reference peptide such asSAP 17.

As mentioned above, the peptide of the present invention is not limitedin its length, and may, for example, comprise up to 100 amino acids oreven more. Irrespective the theory behind the molecular mechanism ofaction, the peptides of the invention have at least 9 preferably 10,more preferably 12, still more preferably 15 or 20, and most preferably10 to 25 or 30 consecutive amino acids derived from said tandem repeat,and wherein said peptides are capable of evoking an immune response in amammal, in particular humans; see also the examples. Since cathepsin L,as mentioned above, furthermore is in the position for a proteolysis atHis-Gly particularly peptides with 17 amino acids are generatedaccording to the invention (i.e. the MUC1 repeat peptide is cleaved attwo sites in one repeat unit (namely at Thr-Ser and His-Gly) whichresults in a fragment of 17 amino acids, see also FIG. 6).

The degradation down to the level of SAP17, however, is inhibited byO-glycosylation at Thr or Ser within the VTSA motif, so thatrespectively glycosylated GVT20 peptides are generated as finalproducts, in particular if O-glycosylation is substantially restrictedto GalNac-residues while longer glycan chains may interfere withprocessing mediated by cathepsin L. In view of the naturally occurringpeptides SAP17 and GVT20, peptides of 17 to 20 amino acids in length areparticularly preferred.

In one embodiment, the peptide according to the invention is a fragmentof said tandem repeat domain. Such fragment can be derived from thetandem repeat domain for example by cleavage with cathepsin L or (an)other enzyme(s) resulting in a peptide according to the invention; seealso infra and the examples.

According to a further embodiment, the invention provides specificpeptides which comprise an amino acid of any one of SEQ ID NOS: 1 to 4or 11, or variants thereof, wherein said variants may comprise one ormore amino acid additions, insertions, substitutions and/or deletions ascompared to the sequence of SEQ ID NOS: 1 to 4 or 11, and wherein thebiological activity, i.e. immunological activity is substantially thesame as the activity of the peptide comprising the unmodified amino acidsequence of SEQ ID NOS: 1 to 4 or 11. In this context, the presentinvention provides the following peptides:

The arrow indicates that the present invention also encompasses variantsof the above mentioned amino acid sequences, which are reduced by one ormore amino acids starting from the C-terminus, under the proviso thatthe variants at least comprise the 9 N-terminal amino acids of the aboveindicated sequences (printed in bold).

The peptides of the present invention can be in their free acid form orthey can be amidated at the C-terminal carboxylate group. The presentinvention also includes analogs of the peptides of the invention. An“analog” of a polypeptide includes at least a portion of thepolypeptide, wherein the portion contains deletions or additions of oneor more contiguous or noncontiguous amino acids, or containing one ormore amino acid substitutions.

“Insertions” or “deletions” are typically in the range of about 1 to 3amino acids. The variation allowed may be experimentally determined bysystematically making insertions, deletions, or substitutions of aminoacids in a polypeptide molecule using recombinant DNA techniques andassaying the resulting recombinant variants for activity. This does notrequire more than routine experiments for the skilled artisan. In caseof MUC1 repeats three positions are known to exhibit a sequencepolymorphism in the population (Engelmann et al., J. Biol. Chem. 276(2001), 27764-27769; international patent application WO00/49045, thedisclosure of which is incorporated in its entirety in this applicationby reference).

Substitutes for an amino acid in the polypeptides of the invention arepreferably conservative substitutions, which are selected from othermembers of the class to which the amino acid belongs. An analog can alsobe a larger peptide that incorporates the peptides described herein. Forexample, it is well-known in the art of protein biochemistry that anamino acid belonging to a grouping of amino acids having a particularsize or characteristic (such as charge, hydrophobicity andhydrophilicity) can generally be substituted for another amino acidwithout substantially altering the structure of a polypeptide. For thepurposes of this invention, conservative amino acid substitutions aredefined to result from exchange of amino acids residues from within oneof the following classes of residues: Class I Ala, Gly, Ser, Thr, andPro; Class II: Cys, Ser, Thr, and Tyr; Class III: Glu, Asp, Asn, and Gln(carboxyl group containing side chains): Class IV: His, Arg, and Lys(representing basic side chains); Class V: Ile, Val, Leu, Phe, and Met(representing hydrophobic side chains); and Class VI: Phe, Trp, Tyr, andHis (representing aromatic side chains). The classes also include otherrelated amino acids such as halogenated tyrosines in Class VI.

Peptide analogs, as that term is used herein, also include modifiedpeptides. Modifications of peptides of the invention include chemicaland/or enzymatic derivatizations at one or more constituent amino acid,including side chain modifications, backbone modifications, and N- andC-terminal modifications including acetylation, hydroxylation,methylation, amidation, and the attachment of carbohydrate or lipidmoieties, cofactors, and the like.

The peptide of the present invention may also comprise one of the groupof D-isomer amino acids, L-isomer amino acids, or a combination thereof.The preparation of peptides comprising D-isomer amino acids is describedfor example in Schumacher, Science 271 (1996), 1854-1857.

The term “biological activity” as used herein is related to theimmunogenic function of the amino acid sequences according to theinvention. As mentioned above, MUC1 is naturally overexpressed invarious cancers, like breast cancer and other adenocarcinomas, andtherefore, it is an important target for immune based anti-cancertherapy. Thus, the MUC1 peptides as disclosed hereinbefore arecontemplated as long as they are capable of inducing an immungenicreaction in mammals, preferably humans, in order to initiate/promote anattack of the patient's immune system against the respective cancer.

The present invention is further directed to a nucleic acid encoding oneof the above mentioned peptides. The term “nucleic acid”, “nucleic acidsequence” and “polynucleotide” are used interchangeably herein and referto a heteropolymer of nucleotides or the sequence of these nucleotides.The polynucleotides of the present invention also include, but are notlimited to, polynucleotides that hybridize to the complement of thedisclosed nucleotide sequences under moderately stringent or stringenthybridization conditions; a polynucleotide which is an allelic variantof any polynucleotide recited above; a polynucleotide which encodes aspecies homologue of any of the herein disclosed proteins; or apolynucleotide that encodes a polypeptide comprising an additionalspecific domain or truncation of the disclosed proteins. Stringency ofhybridization, as used herein, refers to conditions under whichpolynucleotide duplexes are stable. As known to those of skill in theart, the stability of duplex is a function of sodium ion concentrationand temperature (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual 2^(nd) Ed. (Cold Spring Harbor Laboratory, (1989)).Stringency levels used to hybridize can be readily varied by those ofskill in the art.

Low stringency hybridization refers to conditions equivalent tohybridization in 10% formamide, 5× Denhart's solution, 6×SSPE, 0.2% SDSat 42° C., followed by washing in 1×SSPE, 0.2% SDS, at 50° C. Denhart'ssolution and SSPE are well known to those of skill in the art as areother suitable hybridization buffers.

Moderately stringent hybridization refers to conditions that permit DNAto bind a complementary nucleic acid that has about 60% identity,preferably about 75% identity, more preferably about 85% identity to theDNA; with greater than about 90% identity to said DNA being especiallypreferred. Preferably, moderately stringent conditions are conditionsequivalent to hybridization in 50% formamide, 5× Denhart's solution,5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS,at 65° C.

High stringency hybridization refers to conditions that permithybridization of only those nucleic acid sequences that form stableduplex in 0.018M NaCl at 65° C. (i.e., if a duplex is not stable in 0.01SM NaCl at 65° C., it will not be stable under high stringencyconditions, as contemplated herein).

Further, nucleic acid hybridization techniques can be used to identifyand obtain a nucleic acid within the scope of the invention. Briefly,any nucleic acid having some homology to a sequence set forth in thisinvention, or fragment thereof, can be used as a probe to identify asimilar nucleic acid by hybridization under conditions of moderate tohigh stringency. Such similar nucleic acid then can be isolated,sequenced, and analyzed to determine whether they are within the scopeof the invention as described herein.

According to a preferred embodiment, the peptides of the presentinvention are O-glycosylated at one or more of the threonines or serinescontained in the sequence. Preferably, the peptides of any one of SEQ IDNOS: 1 to 4 or 11 are glycosylated at Thr 5 and/or 12. However, also allother serines or threonins in the respective sequences may beglycosylated. A preferred glycan used herein is GalNAc or furthercomplex glycans, which are derived therefrom.

According to a further aspect, the present invention provides a methodof producing the peptides according to the invention, comprising thefollowing steps:

-   (a) providing a peptide comprising the tandem repeat domain of MUC1    or a part thereof, which part at least contains one repeating unit    of said tandem repeat domain of MUC1;-   (b) contacting the peptide of (a) with an effective amount of    cathepsin-L or a closely related enzyme hereof, thereby cleaving the    peptide; and-   (c) isolating the fragments produced in (b).

Preferably, the peptide provided in (a) is a MUC1 protein showing anatural glycosylation pattern. As mentioned above, it was surprisinglyfound in accordance with the present invention that a cathepsin-Lcleavage as performed in step (b), leaves the glycosylation pattern ofthe MUC1 protein, provided in (a), intact. Intact O-glycosylation onprocessed MUC1 repeat peptides in turn contributes to a greater varietyof the MHC class II-restricted helper T cell responses, therebyenhancing an overall anti-tumor response in patients. Thus, the methodof the invention leads to a MUC1 peptide, which can be easily processedby the patient's APCs, for example dendritic cells, by the MHC class IIpathway, and will be presented with an intact glycosylation patternleading to an enhanced immune response of helper T-cells. In thiscontext, it should be noted that there is no restriction regarding theglycosylation pattern, however, threonin glycosylated at the cleavagesite leads to a Thr-Ser bonding, which is stable to cathepsin Lproteolysis. Glycosylation at other sites does not disturb the cleavageaccording to the invention by cathepsin L, but a multipleGal-GalNAc-substitution as well as a substitution with complex glycansmay hamper or even inhibit a fragmentation at His-Gly.

Of course, the above mentioned method is not the only one which leads tosaid peptides, whether glycosylated or not. It is also possible tochemically synthesize those peptides thereby providing, for example, adesired glycosylation pattern. To synthesize glycopeptides,glycosylamino acid building blocks are required which already containthe oligosaccharide chain and threonine or serine. The syntheses ofthese building blocks have been described (Mathieux et al., J. Chem.Soc., Perkin Trans. 1 (1997), 2359-2368). The multiple column solidphase synthesis can be carried out in a semi-manual 20-column multiplesynthesizer, and Wang resin can be selected as support material. TheWang resin (2.5 g) can for example be placed in a glass reactor, swelledin dichloromethane (15 cm³, 10 min.) and washed. A mixture ofFmoc-Ala-OH (3,40 mmol), 1-(mesitylenesulfonyl)-3-nitro-1,2,4-triazole(3,40 mmol) and methylimidazole (3,40 mmol) in dichloromethane (15 cm³)was added. After 2 h, the resin can be washed and the unchanged aminogroups can be acetylated with Ac₂O/DMF (1:1; 15 cm³). The derivatizedresin is then packed for the glycopeptide synthesis in the 20 columns ofthe synthesizer. The reaction and washing solvent can be DMF, the Fmocdeprotections were performed by treatment with piperidine (20%) in DMF(20 min.). The amino acids are coupled as Fmoc amino acid Pfp ester withDhbt-OH (3 mol equiv.). The Gal(1→3)GalNAc-containing building block arecoupled with TBTU and N-ethyldiisopropylamine (1.5 mol equiv.). After 20h reaction time the synthesis cycle is repeated to complete the assemblyof each glycopeptide. After removal of the last Fmoc groups, the resinsare washed, dried, treated with 95% aq TFA (2 cm³, 2 h), and filteredoff. Then, the compounds is treated with catalytic amounts of 1% CH₃ONain methanol at pH 8.5 to remove the acetylic groups of the saccharidepart, and purified by preparative RP-HPLC. The pure O-glycopeptides areobtained in yields of 16-57% after lyophilization. Preferably,glycopeptides are formed containing O-linked GalNAc or elongated complexglycans at one or several of the threonine or serine residues.

The peptides of the invention may also be synthesized by the solid phasemethod using standard methods based on either t-butyloxycarbonyl (BOC)or 9 fluorenylmethoxy-carbonyl (FMOC) protecting groups. Thismethodology is described by G. B. Fields et al. in Synthetic Peptides: AUser's Guide, W. M. Freeman & Company, New York, N.Y. (1992), 77-183.The present peptides may also be synthesized via recombinant techniqueswell known to those skilled in the art. For example, U.S. Pat. No.5,595,887 describes methods of forming a variety of relatively smallpeptides through expression of a recombinant gene construct coding for afusion protein which includes a binding protein and one or more copiesof the desired target peptide. After expression, the fusion protein isisolated and cleaved using chemical and/or enzymatic methods to producethe desired target peptide.

Preferably, the peptide provided in step (a) is represented by naturalMUC1 derived from human milk fat membranes (see Müller et al., J. Biol.Chem. 272 1997, 24780-24793), from tumor ascites (Beatty et al., Clin.Cancer Res. 7 (2001), 781-787) or from human breast carcinoma cell lines(Müller et al., J. Biol. Chem. 277 (2002), 26103-26112) or isrepresented by any one of SEQ ID NOS: 5, 6, 9 or 10 or 12.

Furthermore, the amino acids of the peptide provided in step (a) of theabove method of producing the peptides of the invention areO-glycosylated, however, provided that the peptide is not glycosylatedat the cleaving site of cathepsin-L. Preferably, one or more of thethreonines or serines of the peptide isolated in (c) are O-glycosylated.

According to a further aspect, a peptide is provided, which isobtainable by the above mentioned methods. The peptides of the presentinvention may be employed in a monovalent state (e.g., free peptide orpeptide coupled to a carrier molecule or structure).

The peptides may also be employed as conjugates having more than one(same or different) peptide bound to a single carrier molecule. Thecarrier molecule or structure may be microbeads, liposomes, biologicalcarrier molecule (e.g., a glycosaminoglycan, a proteoglycan, albumin, orthe like), a synthetic polymer (e.g., a polyalkyleneglycol or asynthetic chromatography support), biomaterial (e.g., a materialsuitable for implantation into a mammal or for contact with biologicalfluids as in an extracorporeal device), or others. Typically, ovalbumin,human serum albumin, other proteins, polyethylene glycol, or the likeare employed as the carrier. Such modifications may increase theapparent affinity and/or change the stability of a peptide. The numberof peptide fragments associated with or bound to each carrier can vary.In addition, as mentioned above, the use of various mixtures anddensities of the peptides described herein may allow the production ofcomplexes that have specific binding patterns in terms of preferredligands.

The peptides can be conjugated to other peptides using standard methodsknown to one of skill in the art. Conjugates can be separated from freepeptide through the use of gel filtration column chromatography or othermethods known in the art.

For instance, peptide conjugates may be prepared by treating a mixtureof peptides and carrier molecules (or structures) with a coupling agent,such as a carbodiimide. The coupling agent may activate a carboxyl groupon either the peptide or the carrier molecule (or structure) so that thecarboxyl group can react with a nucleophile (e.g., an amino or hydroxylgroup) on the other member of the peptide conjugate, resulting in thecovalent linkage of the peptide and the carrier molecule (or structure).

As another example, peptides may be coupled to biotin-labeledpolyethylene glycol and then coupled to avidin containing compounds. Inthe case of peptides coupled to other entities, it should be understoodthat the designed activity may depend on which end of the peptide iscoupled to the entity.

Accordingly, in another aspect the present invention relates to a fusionmolecules, also referred to herein as peptide conjugates, comprising apeptide of the invention.

The invention is further directed to an ex vivo-method of producing apopulation of autologous antigen presenting cells (APCs), which arecapable of inducing effective immune responses against MUC1, comprisingthe steps of

-   (a) providing autologous APCs from a tumor patient;-   (b) contacting the autologous APCs from the tumor patient with an    effective amount of a peptide or fusion molecule of the invention    under conditions which allow endocytosis, processing and MHC class    II presentation of the peptides by said APCs; and-   (c) isolating said peptide presenting APCs for the purpose of    immunotherapeutic application in the patient.

Preferably, the MUC1 peptides in (a) are bound to coated ferric oxidebeads. However, it is noted that all other known beads or other carriersand/or conjugates known in the art can be used for the purpose of theabove mentioned method. Generally, all beads can be used, which are notlarger than approx. 1-2 μm in size and allow a covalent coupling ofantibodies and lectines.

Furthermore, an ex vivo-method of producing genetically engineered APCsis provided, which are capable of inducing effective immune responsesagainst MUC1, comprising the steps of:

-   (a) providing a nucleic acid, which encodes one of the peptides or    the fusion molecule of the invention;-   (b) transfecting the APCs with said nucleic acid; and-   (c) selecting APCs, which present said peptides in an MHC II    restricted manner.

According to a preferred embodiment, the nucleic acid in step (a) isprovided in an expression vector. This expression vector preferablycomprises one or more regulatory sequences. The term “expression vector”generally refers to a plasmid or phage or virus or vector, forexpressing a polypeptide from a DNA (RNA) sequence. An expression vectorcan comprise a transcriptional unit comprising an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, promoters or enhancers, (2) a structural or coding sequencewhich is transcribed into mRNA and translated into protein, and (3)appropriate transcription initiation and termination sequences.Structural units intended for use in yeast or eukaryotic expressionsystems preferably include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it may include an N-terminal methionine residue. This residue may or maynot be subsequently cleaved from the expressed recombinant protein toprovide a final product.

According to a further aspect of the invention, an APC is provided,which is obtainable by one of the aforementioned methods. Preferably,this APC is a dendritic cell or a B cell.

Furthermore, the present invention provides a therapeutic orpharmaceutical composition, comprising the peptide, nucleic acids,vectors, fusion molecule and/or the APCs of the invention and apharmaceutically acceptable carrier. Such a composition may also contain(in addition to the ingredient and the carrier) diluents, fillers,salts, buffers, stabilizers, solubilizers and other materials well knownin the art. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. The therapeuticcomposition may further contain other agents which either enhance theactivity or use in treatment. Such additional factors and/or agents maybe included in the therapeutic composition to produce a synergisticeffect or to minimize side-effects. For example, for parenteraladministration, isotonic saline is preferred. For topicaladministration, a cream, including a carrier such as dimethylsulfoxide(DMSO), or other agents typically found in topical creams that do notblock or inhibit activity of the peptide, can be used. Other suitablecarriers include, but are not limited to alcohol, phosphate bufferedsaline, and other balanced salt solutions.

The formulations may be conveniently presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Preferably, such methods include the step of bringing the active agentinto association with a carrier that constitutes one or more accessoryingredients.

Techniques for formulation and administration of the compounds of thepresent application may be found in “Remington's PharmaceuticalSciences”, Mack Publishing Co., Easton, Pa., latest edition.

The compositions contain a therapeutically effective dose of therespective ingredient. A therapeutically effective dose refers to thatamount of the compound sufficient to result in amelioration of symptoms,e.g., treatment, healing, prevention or amelioration of such conditions,specifically in an induction of an immune response in the patient.Suitable routes of administration may, for example, include parenteraldelivery, including intramuscular and subcutaneous injections, as wellas intrathecal, direct intraventricular, intravenous, intraperitonealinjections. Intravenous administration to the patient is preferred.

A typical composition for intravenous infusion can be made up to contain250 ml of sterile Ringer's solution, and 10 mg of the ingredient; seeRemington's Pharmaceutical Science (15^(th) Ed., Mack PublishingCompany, Easton, Pa., 1980). Preferably, the therapeutic composition ofthe present invention is a vaccine. As mentioned above, this vaccinefinds application for use in the treatment of breast cancer or otherMUC1-positive carcinomas including colorectal, pancreatic and gastriccarcinomas.

The present invention is furthermore directed to the use of thepeptides, the nucleic acids, the fusion molecule and/or the APC's ofaccording to the invention for the preparation of a pharmaceuticalcomposition for the treatment of MUC1-positive carcinomas. Thesecarcinoma include breast, colorectal, pancreatic and gastric cancer asmentioned herein before. The agents of the present invention arepreferably formulated in pharmaceutical compositions and thenadministered to a patient, such as a human patient, in a variety offorms adapted to the chosen route of administration. The formulationsinclude, but are not limited to, those suitable for oral, rectal,vaginal, topical, nasal, ophthalmic, or parental (includingsubcutaneous, intramuscular, intraperitoneal, intratumoral, intraorgan,intraarterial and intravenous) administration.

Formulations suitable for parenteral administration conveniently includea sterile aqueous preparation of the active agent, or dispersions ofsterile powders of the active agent, which are preferably isotonic withthe blood of the recipient. Absorption of the active agents over aprolonged period can be achieved by including agents for delaying, forexample, aluminum monostearate and gelatin.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as tablets, troches, capsules,lozenges, wafers, or cachets, each containing a predetermined amount ofthe active agent as a powder, or granules, as liposomes containing theactive agent, or as a solution or suspension in an aqueous liquor ornon-aqueous liquid such as a syrup, an elixir, an emulsion, or adraught. Such compositions and preparations typically contain at leastabout 0.1 wt-% of the active agent. The amount of peptide (i.e., activeagent) is such that the dosage level will be effective to produce thedesired result in the patient.

Aerosol formulations such as nasal spray formulations include purifiedaqueous or other solutions of the active agent with preservative agentsand isotonic agents. Such formulations are preferably adjusted to a pHand isotonic state compatible with the nasal mucous membranes.Formulations for rectal or vaginal administration may be presented as asuppository with a suitable carrier.

In addition, the invention relates to a method of treatment of patientssuffering from a MUC1-positive carcinoma, wherein the therapeuticcomposition described above is administered to the patient in an amounteffective to induce an immune response against MUC1. The appropriateconcentration of the therapeutic agent might be dependent on theparticular agent. The therapeutically effective dose has to be comparedwith the toxic concentrations; the clearance rate as well as themetabolic products play a role as do the solubility and the formulation.Therapeutic efficacy and toxicity of compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED50 (the dose therapeutically effective in 50% of thepopulation) and LD50 (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD50/ED50.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” may be utilized, which ishosted by the National Center for Biotechnology Information and/or theNational Library of Medicine at the National Institutes of Health.Further databases and web addresses, such as those of the EuropeanBioinformatics Institute (EBI), which is part of the European MolecularBiology Laboratory (EMBL) are known to the person skilled in the art andcan also be obtained using internet search engines. An overview ofpatent information in biotechnology and a survey of relevant sources ofpatent information useful for retrospective searching and for currentawareness is given in Berks, TIBTECH 12 (1994), 352-364.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. The above disclosure generallydescribes the present invention. Several documents are cited throughoutthe text of this specification. The contents of all cited references(including literature references, issued patents, published patentapplications as cited throughout this application and manufacturer'sspecifications, instructions, etc) are hereby expressly incorporated byreference; however, there is no admission that any document cited isindeed prior art as to the present invention. In case of conflict, thepresent specification, including definitions, will control.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples intended to limit the scope of the invention.

EXAMPLES

The examples which follow further illustrate the invention, but shouldnot be construed to limit the scope of the invention in any way.Detailed descriptions of conventional methods, such as those employedherein can be found in the cited literature; see also “The Merck Manualof Diagnosis and Therapy” Seventeenth Ed. ed by Beers and Berkow (Merck& Co., Inc. 2003).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art.

Methods in molecular genetics and genetic engineering are describedgenerally in the current editions of Molecular Cloning: A LaboratoryManual, (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory Press); DNA Cloning, Volumes Iand II (Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984);Nucleic Acid Hybridization (Hames and Higgins eds. 1984); TranscriptionAnd Translation (Hames and Higgins eds. 1984); Culture Of Animal Cells(Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors forMammalian Cells (Miller and Calos, eds.); Current Protocols in MolecularBiology and Short Protocols in Molecular Biology, 3rd Edition (Ausubelet al., eds.); and Recombinant DNA Methodology (Wu, ed., AcademicPress). Gene Transfer Vectors For Mammalian Cells (Miller and Calos,eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.154 and 155 (Wu et al., eds.); Immobilized Cells And Enzymes (IRL Press,1986); Perbal, A Practical Guide To Molecular Cloning (1984); thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.);Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker,eds., Academic Press, London, 1987); Handbook Of ExperimentalImmunology, Volumes I-IV (Weir and Blackwell, eds., 1986). Reagents,cloning vectors, and kits for genetic manipulation referred to in thisdisclosure are available from commercial vendors such as BioRad,Stratagene, Invitrogen, and Clontech. General techniques in cell cultureand media collection are outlined in Large Scale Mammalian Cell Culture(Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media(Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian CellCulture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Cultureof Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251);Extracting information from cDNA arrays, Herzel et al., CHAOS 11,(2001), 98-107.

Example 1 Processing of MUC1 by Human Dendritic Cells is Site-Specific

Isolation and Cultivation of Dendritic Cells

Peripheral blood mononuclear cells (PBMCs) from healthy donors wereobtained by leukapheresis followed by Ficoll-density centrifugation.CD14⁺ cells were positively selected using CD14-Microbeads and MACSseparation (Miltenyi Biotech, Bergisch Gladbach, Germany) andsubsequently cultured for 8 days in CellGro Medium (Cellgenix, Freiburg,Germany) supplemented with 800 U/ml of granulocyte-macrophagecolony-stimulating factor (GM-CSF; Sandoz, Basel, Switzerland) and 500IU/ml of IL-4 (CellGenix) at 37° C. and 5% CO₂. GM-CSF and IL-4 werereplenished on days 3 and 5 of culture.

Immortalized dendritic cells (clone D2.4) from C57BL/6 mice were grownin DMEM supplemented with 10% FCS, L-glutamine, 0.1% 2-mercaptoethanol,and antibiotics at 37° C. and 5% CO₂ (Shen et al., J. Immunol. 158(1997), 2723-2730).

MUC1 Glycoforms

Native MUC1 glycoforms were isolated from human tumor ascites (Beatty etal., Clin. Cancer Res. 7 (2001), 781-787) or from human milk fat globulemembranes as described previously. A partially deglycosylated derivativeof the lactation-associated glycoform was generated by treatment withtrifluoromethane sulfonic acid for 30 min at 0° C. (Müller et al., J.Biol. Chem. 272 (1997), 24780-24793). Recombinant fusion proteincontaining six MUC1 repeats was isolated from the cell culturesupernatants after expression in the embryonic kidney cell line EBNA-293as described earlier (Müller and Hanisch, J. Biol. Chem. 277 (2002),26103-26112).

Generation of Synthetic MUC1 Glycopeptides

Glycopeptides H1 to H6 corresponding to MUC1 tandem repeat peptidesbased on the AHG21 sequences AHGVTSAPDTRPAPGSTAPPA (H1 to H3) andAHGVTSAPESR PAPGSTAPAA (H4 to H6) and carrying GalNAc at Thr5, Thr10, orThr17 were chemically synthesized-according to previously-publishedprotocols (Karsten et al., Cancer Res. 58 (1998), 2541-2549) and kindlyprovided by Prof. Hans Paulsen (Institute of Organic Chemistry,University of Hamburg, Germany). The same holds true for glycopeptide A3(substituted with Galβ1-3GalNAc at Thr17), which is based on the samepeptide sequence as H1 to H3. The 100mer peptide corresponding to fiverepeats of the MUC1 domain and starting with the HGV motif wassynthesized by a local facility (University of Pittsburgh) and in vitroglycosylated with GalNAc using purified polypeptideGalNAc-transferases-T1 and -T2 (kindly provided by Dr. Henrik Clausen,School of Dentistry, University of Copenhagen, Denmark) under conditionsdescribed previously (Hanisch et al., J. Biol. Chem. 274 (1999),9946-9954; Hanisch et al., Glycobiology 11 (2001), 731-740). TAP25 andGST20-AES were synthesized in a local facility at the Institute ofBiochemistry (Cologne, Germany).

Antigen Pulse of Dendritic Cells

Human immature DCs were pulsed with native, N-terminally unmodified,soluble antigens, while the mature mouse DCs had to be fed withparticulate antigen to reach sufficient antigen load. Humanmonocyte-derived immature DCs (10⁷ cells in 5 ml Cellgro medium) werepulsed in 6-well cell-culture plates Munc, Wiesbaden, Germany) byincubation with 20 μg/ml soluble antigen (native mucin from tumorascites, 100mer peptide) for a period of 24 h. Simultaneously, thematuration process of the cells was induced by addition of 20 ng/mltumor necrosis factor (TNF-α; Sigma-Aldrich, Munich, Germany) and 10μg/ml of anti-CD40 antibody (Pharmingen, San Diego, Calif.). The cellswere finally separated from medium by centrifugation, washed in PBS andboth fractions were analysed for the presence of peptide/glycopeptidefragments according to the protocol described below.

Mouse dendritic cells DC2.4 (10⁷ cells/1 to 10 ml) were transferred intoa 15 ml Falcon tube, suspended in AIMV medium and preincubated for 1 hat 37° C. (5% CO₂). Antigens were added as native MUC1 (100 μg fromtumor ascites or milk fat globule membranes), as recombinant fusionprotein (100 μg), a 100mer repeat peptide (100 μg) or as a mixture ofbiotinylated glycopeptides H1 to H6 (50 μg) after conjugation toanti-MUC1 antibody (B27.29)-coated dynabeads (each at 5×10⁷ beads/mlfinal concentration). The 1 ml suspension was incubated with occasionalshaking at 37° C. (5% CO₂) for a total time period of 4 h. After pulsingthe cells were separated from the medium by centrifugation (180 g, 5min). The cell fraction was washed several times in phosphate (4 mM),NaCl (153 mM), pH 7.2, while the cell-free supernatant wasre-centrifuged at 3000 g (5 min, 4° C.).

Isolation of Peptides

The human or mouse dendritic cell fractions were treated on ice for 15min with 100 μl 1% NP40, 10 mM Tris-HCl, 150 mM NaCl, pH 8.0 containinga cocktail of protease inhibitors (Sigma P8340, München, Germany)followed by ultrasonication for 2 min. Isolation of MUC1-derived(glyco)peptides was performed in parallel alternative ways: 1) byaffinity chromatography on anti-MUC1 (BW835, C595) antibody columns; 2)by solid-phase extraction on polysphere C18 columns or on Poros 20 R2beads (PerSeptive Biosystems, Framingham, USA), and 3) by binding tostreptavidin-coated magnetic beads (Dynal). To avoid selection ofproduct subfractions by affinity-isolation, in most experiments thepeptides were enriched by solid-phase extraction on reversed-phasecolumns. Due to high antigen load of the cells this non-selectiveenrichment of peptides was sufficient to detect MUC1-specificproteolysis products by mass spectrometry in the presence of cellularbackground.

Anti-MUC1 columns with a total of 1 mg immobilized antibodies wereprepared using HighTrap-NHS columns from Amersham-Pharmacia according tothe manufacturer's instructions. Antibody BW835 was kindly supplied byBehring-Werke, (Marburg, Germany). The cell extracts were cycled twiceover the PBS equilibrated columns at a flow rate of 6 ml per hour in thecold and bound peptides were eluted with 0.1% TFA. Alternatively, thecell extracts were diluted twofold with PBS and incubated with 2×10⁸streptavidin-coated dynabeads M-270 for 30 min at 37° C. and another 30min period with rolling at ambient temperature. After magneticseparation and washing of the beads for three times the beads weretreated with 10 mM dithiothreitol at 56° C. (30 min), and the driedeluate was taken up in 0.1% aqueous trifluoroacetic acid (TFA).Considerable amounts of non-tagged MUC1 glycopeptides were demonstratedto bind to streptavidin-polystyrene-coated dynabeads and to elute duringheating under reducing conditions.

Peptides and glycopeptides contained in the cell-free supernatants wereaffinity-isolated by solid-phase extraction on reversed-phase supports(100 μg polysphere C18, 50 μl Poros C18).

After activation (80% ACN/0.1% TFA), equilibration (water) and loadingof the reversed-phase column with 0.5 to 2 ml of supernatant, the samplewas desalted by washing with water and eluted with 80% acetonitrile(ACN) in 0.1% aqueous TFA.

Mass Spectrometric Analyses

MALDI mass spectrometry: The peptide and glycopeptide samples (20 μl)contained in 0.1% aqueous TFA or in mixtures with acetonitrile wereapplied to the stainless steel target by mixing a 1 μl aliquot with thesame volume of matrix (saturated solution of α-cyano-4-hydroxycinnamicacid in ACN/0.1% TFA, 2:1). Mass spectrometric analysis was performed ona Bruker-Reflex IV instrument (Bruker-Daltonic, Bremen, Germany) bypositive ion detection in the reflectron mode. Ionization ofco-cristallized analytes was induced with a pulsed nitrogen laser beam(337 nm) and the ions were accelerated in a field of 20 kV and reflectedat 23 kV (Hanisch et al., Glycobiology 11 (2001), 731-740; Müller etal., J. Biol. Chem. 274 (1999), 18165-18172).

Nanoflow liquid chromatography with on-line ESI mass spectrometry: LC/MSdata were acquired on a Q-Tof 2 quadrupole-time of flight massspectrometer (Micromass, Manchester, UK) equipped with a Z spray source.Samples were introduced using the Ultimate nano-LC system (LC Packings,Amsterdam, Netherlands) equipped with the Famos autosampler and theSwitchos column switching module. The column setup comprised a 0.3 mm×1mm trap column and a 0.075×150 mm analytical column, both packed with 3μm PepMap C18 (LC Packings, Amsterdam, Netherlands). Samples werediluted 1:10 in 0.1% TFA. 10 μl were injected onto the trap column anddesalted for 3 min using 0.1% TFA and a flow rate of 30 μl/min. The 10port valve switched the trap column into the analytical flowpath and thepeptides were eluted onto the analytical column using a gradient of 5%ACN in 0.1% formic acid to 40% ACN in formic acid over 20 min and acolumn flow rate of approximately 200 nl/min, resulting from a 1:1000split of the 200 μl/min flow delivered by the pump. Survey scans of 1sec covered the range from m/z 400 to m/z 1200. Doubly and triplycharged ions rising above a given threshold were selected for MS/MSexperiments. In MS/MS mode the mass range from m/z 40 to m/z 1400 wasscanned in 1 sec and 10 scans were added up for each experiment. Doublyand triply charged ion masses were deconvoluted using the MaxEndsoftware and the b- and y-ion series were assigned.

Human monocyte-derived immature DCs have previously been studied fortheir ability to take up soluble MUC1 peptide antigen bymacropinocytosis and demonstrated to reach maximum levels ofincorporation within 2 hours (Vlad et al., J. Exp. Med. 196 (2002),1435-1446). Antigen uptake over a period of 24 h was not affected byparallel induction of the maturation process with TNFα and anti-CD40.

Human CD1a⁺ CD14⁻ CD83⁻ dendritic cells were pulsed with native mucinfrom tumor ascites or 100mer peptide either as soluble antigen or asantibody complex. The antibody C595 complex of 100mer peptide was notmore efficiently incorporated and processed by the cells than freeantigen according to quantitative HPLC measurement of 100mer peptide andderived proteolytic fragments in the culture supernatants. In case of100mer peptide, a fraction of the antigen (below 5%) was processed andthe proteolytic products were detected in the cell lysates as well as inthe culture supernatants. Peptide fragments registered by positive ionMALDI(tof) mass spectrometry in the mass range from 1 to 3 kDa weredetected at m/z 1628.7 (SAP17), 1886.7 (GVT20), 2144.9 (GVT23), and2548.0 (STA27) (FIG. 1) and identified by LC-ESI-MS/MS (not shown). Nofragmentation of antigen was revealed after pulsing of DCs with nativeMUC1 from tumor ascites according to mass spectrometric analyses ofcellular lysate or culture supernatant in the mass range up to 8 kDa.All supernatants were checked for the absence of secreted cathepsinL-related activities by incubation of TAP25 peptide and A3 glycopeptidefor 24 h at 37° C. and mass spectrometric analysis of solid-phaseextracted fractions.

Example 2 Site-Specific Processing of MUC1 by Mouse Dendritic Cells isControlled by O-Linked Glycans

Preparation of Bead-Coated Antigen

In some experiments, in which mature mouse DCs were pulsed, a selectedpanel of glycopeptides was non-covalently conjugated to antibody-coatedbeads. Glycopeptides H1 to H6 (100 μg each) were used unmodified orbiotinylated with [2-(biotinamido) ethylamido]-3,3′-dithiopropionic acidN-hydroxysuccinimide ester (Sigma, München, Germany; 100 mM in DMSO, 100μl) at 50° C. over a period of 48 h. After evaporation of the solvent byvacuum centrifugation the biotinylated products were separated fromnon-tagged glycopeptides and excessive reagent by reversed-phasechromatography on a PLRP-S column (Polymer Laboratories, Shropshire,UK). Anti-MUC1 dynabeads were prepared by covalent coupling of 50 μgB27.29 monoclonal antibody (Biomira, Edmonton, Canada) totosyl-activated M-280 beads (Dynal, Hamburg, Germany) in 0.1 M boratebuffer, pH 9.5 (200 μl) for 48 h at ambient temperature. Lectin-coateddynabeads were prepared similarly by conjugation of 50 μg Helix pomatiaagglutinin to M-280 beads. Antibody- and lectin-coated beads (10⁸) werecomplexed with glycopeptides (50 μg) by incubation in 250 μl AIMV mediumunder rolling for 2 h at ambient temperature.

Confocal Laser Scanning Microscopy and Fluorescence-Activated CellSorting

Antigen uptake was quantitated by flow cytometric analysis using aBecton Dickinson FACScalibur according to a previously publishedprotocol (Hiltbold et al., Cell. Immunol. 194: 143-149, 1999). Prior tomicroscopic inspection-DCs were fixed with 2% formaldehyde, andpermeabilized with 0.1% saponin. Following staining with anti-MUC1antibody (B27.29, Biomira, Edmonton, Canada), biotinylated secondaryanti-mouse Ig (Dako, Hamburg, Germany) and FITC-labelled streptavidine(Sigma), the cells were fixed a second time with 1% paraformaldehyde,the chambers of the slides were removed, and the slides were mounted forthe analysis by confocal laser scanning microscopy on a Leica DM IRE2(Hiltbold et al., J. Immunol. 165 (2000), 3730-3741).

The mouse cell line DC2.4 representing mature dendritic cells is knownto have low capacities for antigen uptake by macropinocytosis orreceptor-mediated endocytosis, but has been reported to incorporateparticle bound antigen very effectively (Shen et al., J. Immunol. 158(1997), 2723-2730). For this reason, processing of MUC1 by mouse DCs wasstudied by using bead-conjugated antigen. Mouse DCs were pulsed withnative MUC1 antigen, recombinant fusion protein, 100mer peptide or witha mixture of biotinylated glycopeptides (H1-H6) conjugated to antibody-and/or lectin-coated beads (Tab. 1). Two fractions, the cell pellet andthe supernatant were analysed for the presence of proteolytic fragmentsby MALDI(tof) mass spectrometry to obtain the mass pattern of thepeptide products (Tab. 1 and FIG. 2) and by nanoflow LC-ESI massspectrometry in the MS/MS mode to get sequence information (FIG. 3).

Native MUC1 samples and the recombinant fusion protein did not yielddetectable amounts of peptide fragments (Tab. 1). On the other hand, thenon-glycosylated pentameric repeat peptide (100mer) and theglycopeptides based on the AHG21 sequence were extensively fragmented(Tab. 1, FIG. 2). The 100mer yielded two major fragments with relativemasses at m/z 1888.0 and 1630.0 corresponding to the GVT20 and SAP17peptides derived from the MUC1 repeat sequence (Tab. 1). The AHG21glycopeptides AHGVTSAPD(E)T(S)RPAPGSTAPP(A)A (substituted with oneGalNAc residue) were identified at m/z 2249.0 and 2223.0, respectively,corresponding to the masses of N-thiopropionylated H1 to H3 (m/z 2249.0)and H4 to H6 (m/z 2223.0). The only products identified were registeredat m/z 1695.7 (P1) and m/z 1669.7 (P2), respectively, corresponding tothe GalNAc containing peptide fragments SAP16. The sequence of the twopeptide products (P1, P2) were confirmed by MS/MS on a Qtof2 instrumentto comprize 16 aa long C-terminal portions of the AHG21 glycopeptides(FIG. 3), P1 SAPDTRPAPGSTAPPA SEQ ID NO: 7 and P2 SAPESRPAPGSTAPAA, SEQID NO: 8

both containing GalNAc at Thr/Ser10 or Thr17 (numbering according to theAHG21 sequence). No SAP16 peptides devoid of GalNAc were registered atm/z 1492 and 1466, respectively, indicating that proteolysis of AHG21with GalNAc at Thr5 adjacent to the cleavage site had not occurred andthat GalNAc had not been removed prior to proteolysis. The five aaN-terminal proteolytic fragment AHGVT (N-thiopropionylated) was notdetected in any of the spectra Control experiments with DC primed AIMVmedia without antigen indicated that no proteolytic activity had beensecreted into the medium, since no endopeptidase cleavage of TAP25peptide was detected after 24 h incubation at 37° C. However, afteradjustment of the supernatant to conditions optimal for cysteinproteases (pH 5.5, 1 mM dithiothreitol) minor exopeptidase cleavage ofthe peptide was registered in the mass spectrum. Hence, the SAP16fragments detected in the supernatants of antigen-pulsed cells can beregarded as cellular products and not as extracellular products ofsecreted proteases. TABLE 1 Cellular processing products of native MUC1and MUC1 glycopeptides in mouse dendritic cells Average mass of peptidefragments Structure (structural Antigen^(a) (repeat number) assignment)100mer (HGVTSAPDTRPAPGSTAPPA)₅ (5) 1629.8 (SAP17) Asc-MUC1 (polymeric) —MFP6 (6) —

H1 AHGVTSAPDTRPAPGSTAPPA (1) —

H2 AHGVTSAPDTRPAPGSTAPPA (1) 1695.6 (SAP16 + HexNAc)

H3 AHGVTSAPDTRPAPGSTAPPA (1) 1695.6 (SAP16 + HexNAc)

H4 AHGVTSAPESRPAPGSTAPAA (1) —

H5 AHGVTSAPESRPAPGSTAPAA (1) 1669.6 (SAP16 + HexNAc)

H6 AHGVTSAPESRPAPGSTAPAA (1) 1669.6 (SAP16 + HexNAc)^(a)Asc-MUC1, MUC1 from pooled human tumor ascites; MFP6; MUC1 fusionprotein expressed in human embryonic kidney cell line EBNA 293 (13);biotinylated glycopeptides H1 to H6 with defined glycosylation sites:

GalNAc

Example 3 In Vitro Proteolysis of Native MUC1 and MUC1 Glycopeptideswith Human Cathepsin L Coincides with Cellular Processing

In Vitro Proteolysis of Native MUC1 and MUC1 (Glyco)Peptides with HumanCathepsins

Human cathepsins L and D were purchased from Sigma (München, Germany)and solubilized in 0.1 M sodium acetate buffer, pH 5.5, containing 1 mMEDTA (cathepsin D) and 1 mM dithiotreitol (cathepsin L). 2-5 units ofenzyme(s) were added to 100 μg of mucin or recombinant fusion protein orto 10 μg of (glyco)peptide substrates in a total volume of 20 μldigestion buffer (see above). The reaction mixtures were incubated at37° C. and 2 μl were withdrawn after 3 h or 24 h and diluted 20 fold in0.1% aqueous TFA prior to MALDI mass spectrometry. In case of nativeMUC1 samples with complex O-glycosylation (MUC1 from tumor ascites,HMFG-MUC1, partially deglycosylated HMFG-MUC1, and MFP6 fusion protein)the digest was desalted by solid-phase extraction (ZipTip C18) and the(glyco)peptides were deglycosylated by β-elimination/ethylaminylation in70% ethylamine at 50° C. as previously described (Müller and Hanisch, J.Biol. Chem. 277 (2002), 26103-26112). Each glycosylated position in thepeptide fragments corresponds to the addition of a 27 u mass increment.

3.1 Monomeric Repeat Peptides and Glycopeptides

To verify the processing data obtained with human and mouse DCs and toconfirm the proposed identity of the preferentially involved protease(s)we performed a series of in vitro digestions with cathepsin L andselected MUC1 protein and (glyco)peptide substrates (Tab. 2, and 3, FIG.4). Using standard conditions for cysteine proteases and incubationtimes of 3 h the enzyme was able to cleave all non-glycosylatedmonomeric MUC1 repeat peptides quantitatively, except for the variantGST20-AES peptide (80% cleavage). Irrespective of the N-terminaltripeptide motif in the repeat sequence (TAP, AHG, GST) and of thelength of the peptides (20mer, 21mer, 25mer), human cathepsin L cleavedspecifically between Thr-Ser in the VTSA motif of the repeat peptide(Tab. 2). Besides this preferential cleavage site, which is inaccordance with the cellular processing, minor activities of the enzymepreparation were found to be directed to the adjacent positions Val-Thr(TSA17 at m/z 1958.8) and Ser-Ala (APD15 at m/z 1771.0). To examine thepossibility that aminopeptidases could be responsible for the generationof these minor products, a protected substrate carrying a biotin labelat the amino terminus was used as substrate (FIG. 4). The terminallyprotected glycopeptide showed only one major product at m/z 1858.7corresponding to the glycosylated SAP16 fragment (FIG. 4C). Catalyticactivity of the cystein endopeptidase directed to the Thr-Ser bond wasspecifically inhibited with 1 μM Leu-Leu-Leu fluoromethyl ketone, whileminor aminopeptidase activity in the cathepsin L preparation remainedunaffected (FIG. 4B). In agreement with DC-mediated processing was thefinding that O-glycosylated peptides, carrying GalNAc or Galβ1-3GalNAc,were effectively digested (Tab. 2, FIGS. 4A,C). However, the position ofglycan attachment to one of the tree threonines (Thr5, Thr10, Thr17) inthe AHG21 sequence was found to be critical as suggested by results fromcellular processing. While GalNAc or Galβ1-3GalNAc in positions moredistant from the cleavage site (Thr10, Thr17) had no influence on thecleavage by cathepsin L, the glycopeptides H1 and H4, both beingglycosylated at the cleavage site (Thr5), were stable to proteolysis.Minor exopeptidase activity was detectable (in case of theseglycopeptides) in the cathepsin L preparation from human liver. TABLE 2In vitro proteolysis of MUC1 glycopeptides and peptides with humancathepsin L Substrate Structure/Sequence Average mass of production(mass range 500-4500 Da) Synthetic monomeric repeat peptides H1

2162.8 (AHG21 + HexNAc) H2

1696.5 (SAP16 + HexNAc) H3

1696.7 (SAP16 + HexNAc) A3

1858.0 (SAP16 + Hex-HexNAc) TAP25 SEQ ID NO: 9

1491.8 (SAP16) GST20-AES SEQ ID NO: 10

910.8 (SAP9)

TABLE 3 In vitro proteolysis of MUC1 glycopeptides and peptides withhuman cathepsin L Substrate Structure/Sequence (repeat number) Averagemass of repeat fragment ion (structural assignment) Native mucinHMFG-MUC1^(a) (polymeric) —^(b) GalNAc-MUC1^(a) (polymeric) 1915.7,1942.7, 1969.7, 1996.8 (GVT20 + 1-4 HexNAc)^(b) Asc-MUC1^(a) (polymeric)—^(b) MFP6^(a) (6) —^(b) Synthetic oligomeric peptides 100mer

1888.0 (GVT20), 1629.5 (SAP17) Tn-100mer

2498.1 (GVT20 + 3 HexNAc) Tf-100mer

—3.2 Mucins and Oligomeric Repeat Peptides and Glycopeptides

Oligomeric MUC1 repeat domains with complex and dense O-glycosylation(native mucin from HMFGs and tumor ascites, and recombinant fusionprotein) were not digested by cathepsin L (Tab. 3). On the other hand,after partial de-O-glycosylation of HMFG-MUC1 by controlled chemicalcleavage of peripheral and backbone sugars (Müller et al., J. Biol.Chem. 272 (1997), 24780-24793) the derivative with residual GalNAcsubstitution revealed fragmentation by cathepsin L at low efficiency(Tab. 3). The products (registered at m/z 1915.7, 1942.7, 1969.7, and1996.8), which were detectable after β-elimination of GalNAc and Michaeladdition of ethylamine (Hanisch et al., Anal. Biochem. 290: 47-59,2001), correspond to 20meric peptides of the MUC1 repeat domain carryingone to four substituents. Sequencing by ESI-MS/MS revealed that the20meric peptide started with the GVT motif. The same peptide product wasdetected on digestion of GalNAc-substituted 100mer (Tn-100mer) carryingthree sugar residues per repeat at each of the threonines. However, thecathepsin L catalyzed fragmentation of Tn-100mer resulted in theformation of only tiny amounts of glycosylated GVT20 (Tab. 3). Using thederived Tf-100mer with two to three corel disaccharides per repeat noproteolytic fragmentation was registered (Tab. 3).

Contrasting to the glycopeptides, the non-glycosylated 100mer peptidewas extensively degraded in vitro resulting in the intermediateformation of GVT20 peptides (dominating after 3 h), which were finallycleaved (during 24 h) at the Thr-Ser bond to yield SAP17 (Tab. 3). Thesefindings point to a general inhibition of proteolytic activity byO-glycosylation being apparent already on the monosaccharide level, buteven more strongly on the disaccharide or higher levels of complexity.

As a control, human cathepsin D was tested with a selected panel of MUC1repeat peptides and glycopeptides and found to be unable to use any ofthese as a substrate, even if incubation times of up to 24 h were chosen(Tab. 4). It can be concluded that proteolytic activity in the humancathepsin L preparation recapitulated all major aspects of MUC1glycopeptide processing in human and mouse DCs. TABLE 4 In vitroproteolysis of MUC1 glycopeptides and peptides with human cathepsin DAverage mass of production Sub- Structure/Sequence (mass range strate(repeat number) 500-4500 Da)

H1 AHGVT-SAPDTRPAPGSTAPPA 2161.9 (AHG21 + HexNAc)

H2 AHGVT-SAPDTRPAPGSTAPPA 2161.9 (AHG21 + HexNAc)

H3 AHGVT-SAPDTRPAPGSTAPPA 2161.9 (AHG21 + HexNAc) 100mer[HGVT-SAPDTRPAPGSTAPPA] (5) —The substrates (10 to 100 μg in 20 μl 0.1 M sodium acetate buffer, pH5,5. containing 1 mM EDTA were incubated with 0 cathepsin D for 24 h orwith cathepsin L for 3 h (in the presence of 1 mM DTT) at 37° C.

refers to O-linked GalNAc,

to O-linked Galβ1-3GalNAc.^(a)HMFG-MUC1, mucin from human milk, fat globule membranes,GalNAc-MUC1. partially deglycosylated HMFG-MUC1; Asc-MUC1, mucin frompooled tumor ascites; MFP6, recombinant fusion protein expressed in thehuman embryonic kidney cell line EBNA-293; H1, H2, H3, A3, TAP25 andGST20-AES represent N-terminally unmodified (glyco)peptides.^(b)Mass spectra were recorded after de-O-glycosylation/ethylaminylation(12).

Example 4 In Vitro Proteolysis of MUC1 Glycopeptides with Enzymes inLow-Density Endosomal Fractions from Mouse Dendritic Cells Coincideswith Cellular Processing

Mouse dendritic cells (10⁸) were homogenized by fine-needle aspirationon ice using 1 ml of 0.3 M sucrose, 0.01 M Hepes as buffer (withoutprotease inhibitors). After dilution to 7 ml and centrifugation at 850 gfor 10 min to remove intact cells and nuclei, 6 ml of the supernatantwere centrifuged over 24 ml of 30% Percoll with 0.3 M sucrose, 0.01 MHepes for 105 min at 20.000 rpm in a centrifuge (model J2-21 M/E, rotor:JA-20, Beckman instruments, München, Germany) (Barnes et al., J. Exp.Med. 181 (1995), 1715-1727). The gradient was fractionated by gravitysiphon (30×1 ml) and each fraction was analysed after sonication for thepresence of MHC class II molecules by enzyme immunoassay with anti-H2antibody (rat hybridoma cell line M1/42.3.9.8.HLK obtained from theATCC), β-hexosamimidase activity (Barnes et al., 1995) and cathepsin Lrelated proteolytic activity using TAP25 peptide as substrate (5 μg).The samples were incubated for 24 h at 37° C., diluted 20 fold inaqueous TFA and analysed by MALDI mass spectrometry. For specificinhibition of cathepsin L activity, the corresponding fractions weremixed with 1 μM Z-Leu-Leu-Leu-fluoromethyl ketone (Sigma). Westernblotanalyses of 290 μl aliquots of the density gradient fractions wereperformed under standard conditions (Müller et al., J. Biol. Chem. 274(1999), 18165-18172) using anti mouse cathepsin L antibody CPLH 3G10(Alexis Biochemicals, Grünberg, Germany).

Mouse dendritic cells were ruptured in the absence of proteaseinhibitors and the supernatant (after removal of nuclei) was centrifugedin a Percoll gradient. The gradient fractions were tested forproteolytic activity using TAP25 as a substrate and incubationconditions were optimized for cysteine proteases (FIG. 5). Low densityendosomes were separated from lysosomes according to the registration ofmarker proteins (β-hexosamimidase) (FIG. 5) and demonstrated to containa cysteine protease inhibitable with Z-Leu-Leu-Leu-fluoromethyl ketoneand with a site-specificity related to human cathepsin L.

Mouse cathepsin L was identified in low density fractions (fractions 22to 30) by westernblot analysis using a monoclonal antibody (FIG. 5,insert). Cathepsin L-like enzymatic activity was isographic with thesepositively stained fractions, since enzymes in fractions with a densityof approx. 1.037 g/ml cleaved TAP25 peptide at Thr-Ser yielding SAP16,while all other fractions, in particular those with densities above1.054 g/ml, contained no such activity, but considerable activities ofcarboxypeptidase(s).

The presented work for the first time reveals insight into the molecularaspects of processing by human and murine DCs of the human glycoproteintumor antigen MUC1, a mucin, which could serve as a model for processingof other heavily O-glycosylated antigens. Using state-of-the-artmethodologies for the structural characterization of (glyco)peptidesthis study was able to answer four important questions regarding MUC1proteolysis by APCs in the MHC class II pathway: 1) Where are thecleavage sites in the MUC1 repeat peptide? 2) In which way do O-linkedglycans affect proteolytic cleavage? 3) Are core-type glycans removedprior to proteolytic processing? 4) Which of the enzymes involved in theprocessing machinery are responsible for proteolytic cleavage of MUC1repeat peptides? The results obtained in accordance with the presentinvention suggest that MUC1 repeats are cleaved mainly at two sites, atthe His-Gly bond and between Thr-Ser in the VTSA motif (FIG. 6). Duringcellular processing the core-type glycans GalNAc and Galβ1-3-GalNAc werenot removed (see also Vlad et al., J. Exp. Med. 196 (2002), 1435-1446,),but inhibited the cleavage if they were located adjacent to the cleavagesite. The revealed molecular aspects, in particular the site specificityof cleavage and the site-dependent effects of carbohydrates coincided inthe human and mouse system, and were in perfect agreement in thecellular and in vitro assays. Moreover, the cellular processing afterpulsing of DCs with biotinylated bead-conjugated glycopeptides was incoincidence with the processing of native, untagged antigen in a solubleform. This series of coincidences reduces the likeliness of artefacts,which might have been introduced by the use of tagged immobilizedantigen in some of the experiments.

The structural features of MUC1 processing products (FIG. 6) matchfindings from an immunological study (Vlad et al., J. Exp. Med. 196(2002), 1435-1446). According to this work, glycans remain intact duringprocessing of MUC1 glycopeptides by DCs, but influence activation of Tcell hybridoma clones in a site-specific manner. Clone VF5, reactive toa peptide epitope that comprizes the DTR motif, was activated by DCspulsed with AHG21 glycopeptides which carried glycans at Ser16 or Thr17.No activation of this clone was measurable, however, if the glycans werelocated at the proposed epitope or at the Thr/Ser positions adjacent tothe cathepsin L cleavage site defined in the present study (Thr5-Ser6).Hence the O-linked glycans can alter proteolytic processing orpresentation of the MHC class II-restricted glycopeptides in asite-specific manner. While glycans alter processing of glycopeptidesthey do not always affect binding of processed glycopeptides by MHCclass II, as was demonstrated previously (Jensen et al, J. Immunol. 158(1997), 3769-3778).

Cathepsin L, a cysteine protease related to papain, has been claimed tobe involved in antigen processing (Nakagawa et al., Immunol. Rev. 172(1999), 121-129; Honey et al., J. Biol. Chem. 276 (2001), 22573-22578).We confirmed the possible involvement of cathepsin L (or a closelyrelated enzyme species) in MUC1 repeat proteolysis by specificinhibition of the human and mouse enzyme. The in vitro data withcathepsin L show that oligomeric tandem repeats are fragmented by theenzyme to intermediate GVT20 peptides (FIG. 6), a process which is notsite-controlled, but quantitatively affected by O-glycosylation.Accordingly, substitution with three GalNAc residues reduces proteolyticactivity, and substitution with three Gal-GalNAc disaccharides resultsin no detectable fragmentation (FIG. 6). The initially formed GVT20peptides are finally degraded to SAP17 under the prerequisite that Thrin the VTSA motif is not substituted with a glycan. This site is,however, a preferred target for the ubiquitously expressed polypeptideGalNAc-transferases, ppGalNAc-T1 and -T2, (which were used for in vitroglycosylation of 100mer peptide). In agreement with this, the Tn-100merand the densely O-glycosylated native mucin samples were characterizedin previous studies (Müller et al., S. Biol. Chem. 272 (1997),24780-24793; Beatty et al., Clin. Cancer Res. 7 (2001), 781-787) tocarry glycans at this site throughout (Tn-100mer) or in the majority ofthe tandem repeats (native MUC1 from cancer cells). Non-glycosylation ofthe cathepsin L cleavage site in two adjacent repeats should,accordingly, be rare and the formation of SAP17 fragments from thesemucin samples unlikely. The highly specific processing of MUC1 and theconcomittant restriction of effective proteolytic cleavage to particularglycoforms of the mucin repeat domain would explain the weakimmunogenicity of native MUC1 from milk fat membranes or from tumorascites as related to the weak T cell responses observed in previousstudy (Vlad et al., J. Exp. Med. 196 (2002), 1435-1446). The masking ofpotential processing sites by O-glycosylation might also be a newmechanism on the level of posttranslational protein modification toavoid autoimmunity against otherwise immunogenic protein backbones.

Although cathepsin L may not be required for the generation of amajority of epitopes it can strongly affect the generation of a subsetof antigenic epitopes in both a positive and a negative fashionsuggesting a direct role for this protease, but also for the relatedcathepsin S, in antigen processing (Hsieh et al., J. Immunol. 168(2002), 2618-2625). It can be anticipated that antigen processing inlate endosomes is mediated by a family of proteases with partiallyoverlapping, but still distinct specificities. Hence, the in vitro dataon cathepsin L cleavage of MUC1 presented in this paper do not excludethe possible involvement of other, cathepsin L related, enzymes in theprocessing machinery and in the specific cleavage of MUC1. Sequence datasuggest that cathepsins L and S are the most closely related enzymes inthis family (Santamaria et al., J. Biol. Chem. 274 (1999), 13800-13809),if a recently identified isoform of cathepsin L, cathepsin L2, is nottaken into account (Santamaria et al., Cancer Res. 58 (1998),1624-1630). Sequence homology and identical cleavage patterns observedin cellular processing by human or mouse DCs and in vitro with humancathepsin L support the assumption that the enzymes in both speciesexhibit similar specificities.

Tumor-associated MUC1, in particular the glycoforms from breast cancercells, have been claimed to exhibit underglycosylated protein cores(Lloyd et al., J. Biol. Chem. 271 (1996), 33325-33334), referring toboth, to truncated chain lengths and to a reduced number of glycosylatedsites per repeat. Recently, it could be shown that this finding cannotbe transferred to secreted mucin, since the structural analysis of MUC1samples that were recombinantly expressed in four different breastcancer cell lines revealed increased substitution densities withcomplex, individually fluctuating O-glycans (Müller et al., J. Biol.Chem. 277 (2002), 26103-26112). An average profile of O-linked glycansdetermined for MUC1 from pooled ascites samples of breast and pancreaticcancer patients was also in accord with a more complex glycosylation(Jensen et al., J. Immunol. 158 (1997), 3769-3778). In agreement withour findings, this glycoform of the mucin represents a weak immunogen inthe MHC class I pathway (Hiltbold et al., Cell. Immunol. 194 (1999),143-149) and is non-immunogenic in the MHC class II pathway (Hiltbold etal., J. Immunol. 165 (2000), 3730-3741). The latter phenomenon has beenassigned to an entrapment in early endosomes of DCs mediated bymultivalent, high-avidity interaction with the mannose receptor(Hiltbold et al., J. Immunol. 165 (2000), 3730-3741). It can beconcluded, accordingly, that O-glycosylation of MUC1 interferes first ofall with trafficking of endocytosed mucin. Later on, if late endosomalcompartments are accessible for the antigen, other modes of interferencemediated by O-linked glycans could also come into play, like inhibitionof proteolysis. The existence of such restrictions introduced bysite-specific O-glycosylation became evident in the present study, sinceglycans linked to Thr/Ser in the VTSA motif of MUC1 repeats preventedprocessing of the glycopeptides (FIG. 6). The site-specificity of glycansubstitution has to be considered in the design of synthetic cancervaccines. To make glycosylated epitopes available for MHC class IIpresentation, it might be advantageous to use synthetic glycopeptideslacking glycosylation at the VTSA motif. Moreover, SAP17 peptides andtheir glycosylated derivatives may represent a “pre-processed” formsuitable for external loading on MHC class II molecules inimmunotherapeutic approaches. In loading experiments with SAP17 theglycosylation-dependent effects on the binding to MHC class II proteinsand on recognition by the T cell receptors can now be studied bysystematic variation of the substitution sites and structures of theglycans.

Hence, the present invention provides a novel approach for the design ofimmunogenic MUC1 peptides that can be used as anti-cancer vaccines.

1. A peptide of least 9 amino acids in length derived from the tandemrepeat domain of MUC1 and having the amino acid sequence SAP at itsN-terminus.
 2. The peptide of claim 1, wherein said peptide comprises of10 to 25 amino acids of said tandem repeat domain of MUC1.
 3. Thepeptide of claim 1 or 2, which is a fragment of said tandem repeatdomain.
 4. The peptide of claim 1, which comprises an amino acidsequence of any one of SEQ ID NOS: 1 to 4 or 11, or variants thereof,wherein said variants comprise one or more amino acid additions,insertions, substitutions or deletions as compared to the sequence ofany one of SEQ ID NOS: 1 to 4 or 11, and wherein the biological activityof said peptide is substantially equal to the activity of the peptidecomprising the unmodified amino acid sequence of any one of SEQ ID NOS:1 to 4 or
 11. 5. The peptide of claim 1, wherein one or more threoninesor serines of the peptide are O-glycosylated.
 6. The peptide of claim 5,having an amino acid sequence of any one of SEQ ID NOS: 1 to 4 or 11,wherein the amino acid is glycosylated at Thr 5 or
 12. 7-24. (canceled)25. The peptide of claim 3, wherein said peptide is SAP17 (SEQ ID NO:11).
 26. A nucleic acid encoding a peptide of claim
 1. 27. A method ofproducing a peptide having at least 9 amino acids derived from thetandem repeat domain of MUC1 and having the amino acid sequence SAP atits N-terminus, comprising the following steps: (a) providing a peptidecomprising the tandem repeat domain of MUC1 or a part thereof, whichpart at least contains one repeating unit of said tandem repeat domainof MUC1; (b) contacting the peptide of (a) with an effective amount ofcathepsin-L or a closely related enzyme hereof, thereby cleaving thepeptide; and (c) isolating the fragments produced in (b).
 28. The methodof claim 27, wherein the peptide provided in step (a) is natural MUC1derived from human milk fat membranes, from human tumor ascites, or fromhuman breast carcinoma cell lines or is represented by any one of SEQ IDNOS: 5, 6, 9, 10, or
 12. 29. The method of claim 27, wherein one or moreof the amino acids of the peptide provided in step (a) isO-glycosylated, provided that the peptide is not glycosylated at thecleaving site of cathepsin-L.
 30. The method of claim 27, wherein one ormore threonines or serines of the peptide fragment isolated in (c) areO-glycosylated.
 31. A peptide obtainable by the method of claim
 27. 32.A fusion molecule comprising the peptide of claim
 31. 33. An exvivo-method of producing a population of autologous antigen presentingcells (APCs), which are capable of inducing effective immune responsesagainst MUC1, comprising the steps of (a) providing autologous APCs froma tumor patient; (b) contacting the autologous APCs from the tumorpatient with an effective amount of a peptide or fusion moleculecomprising at least 9 amino acids of the tandem repeat domain of MUC1and having the amino acid sequence SAP at its N-terminus, wherein saidcontacting is under conditions which allow endocytosis, processing, andMHC class II presentation of fragments of said peptide or fusionmolecule by said APCs; and (c) isolating said peptide or fusion moleculefragment-presenting APCs for the purpose of immunotherapeuticapplication in the patient.
 34. The method of claim 33, wherein saidpeptide or fusion molecule comprises 10 to 25 amino acids of said tandemrepeat domain of MUC1.
 35. The method of claim 33, wherein said peptideor fusion molecule is a fragment of said tandem repeat domain.
 36. Themethod of claim 35, wherein said peptide or fusion molecule is SAP17(SEQ ID NO: 11).
 37. The method of claim 33, wherein said peptide orfusion molecule comprises an amino acid sequence of any one of SEQ IDNOS: 1 to 4 or 11, or variants thereof, wherein said variants compriseone or more amino acid additions, insertions, substitutions or deletionsas compared to the sequence of any one of SEQ ID NOS: 1 to 4 or 11, andwherein the biological activity of said peptide or fusion molecule issubstantially equal to the activity of the peptide or fusion moleculecomprising the unmodified amino acid sequence of any one of SEQ ID NOS:1 to 4 or
 11. 38. The method of 33, wherein one or more threonines orserines of said peptide or fusion molecule are O-glycosylated.
 39. Themethod of claim 38, wherein said peptide or fusion molecule has an aminoacid sequence of any one of SEQ ID NOS: 1 to 4 or 11, wherein the aminoacid is glycosylated at Thr 5 or
 12. 40. The method of claim 33, whereinthe peptides or fusion molecules in (b) are bound to ferric oxide beads.41. An ex vivo-method of producing genetically engineered antigenpresenting cells (APCs), which are capable of inducing effective immuneresponses against MUC1, comprising the steps of (a) providing a nucleicacid encoding a peptide or fusion molecule comprising at least 9 aminoacids of the tandem repeat domain of MUC1 and having the amino acidsequence SAP at its N-terminus, (b) transfecting the APCs with saidnucleic acid, and (c) selecting APCs, which present said peptides in anMHC II restricted manner.
 42. The method of claim 41, wherein thenucleic acid of step (a) is provided in an expression vector.
 43. Themethod of claim 41, wherein said peptide comprises 10 to 25 amino acidsof said tandem repeat domain of MUC1.
 44. The method of claim 41,wherein said peptide is a fragment of said tandem repeat domain.
 45. Themethod of claim 41, wherein said peptide is SAP17 (SEQ ID NO: 11). 46.The method of claim 41, wherein said peptide comprises an amino acidsequence of any one of SEQ ID NOS: 1 to 4 or 11, or variants thereof,wherein said variants comprise one or more amino acid additions,insertions, substitutions or deletions as compared to the sequence ofany one of SEQ ID NOS: 1 to 4 or 11, and wherein the biological activityof said peptide is substantially equal to the activity of the peptidecomprising the unmodified amino acid sequence of any one of SEQ ID NOS:1 to 4 or
 11. 47. The method of 41, wherein one or more threonines orserines of said peptide are O-glycosylated.
 48. The method of claim 47,wherein said peptide has an amino acid sequence of any one of SEQ IDNOS: 1 to 4 or 11, wherein the amino acid is glycosylated at Thr 5 or12.
 49. An antigen presenting cell (APC) obtainable by the method ofclaim
 41. 50. The APC of claim 49, which is a dendritic cell or a Bcell.
 51. A composition comprising a therapeutically effective amount ofa peptide or fusion molecule comprising at least 9 amino acids of thetandem repeat domain of MUC1 add having the amino acid sequence SAP atits N-terminus or an antigen presenting cell comprising said peptide orfusion molecule and a pharmaceutically acceptable carrier.
 52. Thecomposition of claim 51, wherein said peptide or fusion moleculecomprises 10 to 25 amino acids of the tandem repeat domain of MUC1. 53.The composition of claim 51, wherein said peptide or fusion molecule isa fragment of said tandem repeat domain.
 54. The composition of claim53, wherein said peptide or fusion molecule is SAP17 (SEQ ID NO: 11).55. The composition of claim 51, wherein said peptide or fusion moleculecomprises an amino acid sequence of any one of SEQ ID NOS: 1 to 4 or 11,or variants thereof, wherein said variants comprise one or more aminoacid additions, insertions, substitutions or deletions as compared tothe sequence of any one of SEQ ID NOS: 1 to 4 or 11, and wherein thebiological activity of said peptide or fusion molecule is substantiallyequal to the activity of the peptide or fusion molecule comprising theunmodified amino acid sequence of any one of SEQ ID NOS: 1 to 4 or 11.56. The composition of 51, wherein one or more threonines or serines ofsaid peptide or fusion molecule are O-glycosylated.
 57. The compositionof claim 56, wherein said peptide or fusion molecule has an amino acidsequence of any one of SEQ ID NOS: 1 to 4 or 11, wherein the amino acidis glycosylated at Thr 5 or
 12. 58. The composition of claim 51, whichis a vaccine.
 59. A method of treating a patient suffering from aMUC1-positive carcinoma, said method comprising administering acomposition comprising a peptide or fusion molecule comprising at least9 amino acids of the tandem repeat domain of MUC1 and having the aminoacid sequence SAP at its N-terminus or an antigen presenting cellcomprising said peptide or fusion molecule, wherein said composition isadministered to said patient in an amount effective to induce an immuneresponse against MUC1.
 60. The method of claim 59, wherein said peptideor fusion molecule comprises 10 to 25 amino acids of the tandem repeatdomain of MUC1.
 61. The method of claim 59, wherein said peptide orfusion molecule is a fragment of said tandem repeat domain.
 62. Themethod of claim 61, wherein said peptide or fusion molecule is SAP17(SEQ ID NO: 11).
 63. The method of claim 59, wherein said peptide orfusion molecule comprises an amino acid sequence of any one of SEQ IDNOS: 1 to 4 or 11, or variants thereof, wherein said variants compriseone or more amino acid additions, insertions, substitutions or deletionsas compared to the sequence of any one of SEQ ID NOS: 1 to 4 or 11, andwherein the biological activity of said peptide or fusion molecule issubstantially equal to the activity of the peptide or fusion moleculecomprising the unmodified amino acid sequence of any one of SEQ ID NOS:1 to 4 or
 11. 64. The method of 59, wherein one or more threonines orserines of said peptide or fusion molecule are O-glycosylated.
 65. Themethod of claim 64, wherein said peptide or fusion molecule has an aminoacid sequence of any one of SEQ ID NOS: 1 to 4 or 11, wherein the aminoacid is glycosylated at Thr 5 or
 12. 66. The method of claim 59, whereinsaid composition is a vaccine.
 67. The method of claim 59, wherein theMUC1-positive carcinoma is a breast, a colorectal, a pancreatic or agastric cancer.